Yatha Ahu Vairyo Cancer-induced cachexia is an under explored problem with lethal consequences. To date specialized nutritional supplements have failed as anti-cachexia treatments. There are no known cures for this condition, and the multi-faceted nature of cachexia makes it difficult to investigate. Our purpose in Aim 1 in this supplemental application is to use mass spectrometry imaging (MSI) and MS to expand our understanding of the metabolic pathways that are altered in pancreatic ductal adenocarcinoma (PDAC) of patients with and without cachexia. These studies may lead to MS-derived plasma markers of cachexia, and potentially of PDAC. The MSI characterization of PDAC will identify potential new targets in the treatment of cachexia. We intend to initially focus on the glutamine-glutamate axis to understand the role of glutamine in cancer-induced cachexia. In Aim 2 we will silence the glutamine transporter ASCT2 or glutaminase (GLS) in an established model of PDAC induced cachexia and determine the impact on weight loss, and tumor and organ metabolism using MSI and 1H magnetic resonance spectroscopy. These studies will expand our understanding of glutamine metabolism in cancers and its role in modifying the metabolism of organs, including the brain. To the best of our knowledge the studies in Aims 1 and 2 have not been previously performed. MSI is a powerful technology that provides spatial information of metabolites and their intermediaries that can be related to immunohistochemical characterization of the tumor section, such as endothelial cells, stromal cells and immune cells. In Aim 1, MSI/MS characterization of PDAC tissue and plasma from patients with and without cachexia will provide a wealth of information of these cancers and an important data base that can continue to be mined in the future. Our 1H MRS results from the parent R01 have identified changes in glutamine and glutamate in tumor interstitial fluid and the brain in tumor xenografts, and in human plasma. The studies in Aim 2 will investigate, for the first time, the effect of silencing the ASCT2 glutamine transporter or silencing GLS in preventing PDAC cells from inducing cachexia. We will identify, for the first time, the impact of these ASCT2 or GLS silenced cells on tumor and body organ metabolism using MSI/MS and high-resolution 1H MRS of extracts. These studies will identify potential therapeutic strategies to treat PDAC induced cachexia. The studies will significantly advance the scope of our parent R01, and induct two outstanding investigators into the cachexia research field.